PCR Practice
Technical Log
Name: HaoQi (Esther) Li
Date: 06/23/06 Friday
Work Location: Naval Medical Research Center, Spring Field, MD
Title of Project: Developing an Optimized qPCR for Borrelia lonestari
1. Objective – Why did you hope to accomplish today?
I hoped to work with the Clustal W program and Gene Doc, and also observe the final
process of the PCR practice.
2. Events – a. summarize the schedule of events. Procedures performed in detail,
results, analysis, with tables, charts
Experiment has not been started today.
b. summarize communications,, emails, conversations, pertinent material read +
reactions
In the morning, I reformatted the Borrelia sequence document to fit the format of
http://www.ebi.ac.uk/clustalw/ and tried the program. However, the program could not
run with all the strands. When I copied and pasted two strands into the program, it
worked.
I discovered that the lab computer does not block out mail.tjhsst.edu so from now on I
can attach all documents to the school’s email account!
Downloading http://www.psc.edu/biomed/genedoc/
Through http://en.wikipedia.org/wiki/DNTP and
http://info.med.yale.edu/genetics/ward/tavi/p13.html I learned that dNTP stands for
“dNucleotideTriPhosphate.”
Dr. Ju took out my three tubes (A, B, NC) and the congealed agarose gel from the 4C
chamber. She placed the gel in the gel electrophoresis apparatus and covered the gel with
the buffer solution. Then Sindhu and I each extrapolated 3 drops of 2l of 5X dye
marker lined up on a strip of film paper. Then I used a pipette to extrapolate 8l of
solution from each of the three tubes (A, B, NC) and mixed each 8l with a drop of the
5X dye by pipetting the solution in and out of the pipette. After the mixture, I carefully
inserted 8l of the solution into a well using both hands and carefully pressing out the
solution. After all six wells have been loaded (three were mine, three were Sindhu’s), I
added 8l of 100basepair DNA Ladder as a standard. This DNA ladder has the smallest
fragment of 100bp and it increases in intervals of 100. 600bp is a double strand just in
case the first 100bp runs off the gel. Then I connected the gel with electric power and set
it at 150V. I saw bubbles forming, which indicating that electricity was indeed running
through.
After thirty minutes, the gel looked like:
Wells
Standard
NC
B
A
NC
B
A
Amplicon bands
only visible under
UV
The purple lines are the 100bp
markers provided by the dye, which
makes sense since the dye in the
100bp standard runs parallel to it.
The dye (which is added to the six
wells) also contains a blue fragment
serving the purpose to mark the
other end of the gel electrophoresis.
Amplicon is the product of PCR, i.e. DNA having been amplified in quantity.
In order to see the Amplicon bands, Dr. Ju took Sindhu and me to another lab with a UV
machine named Gel Doc 2000. Dr. Ju placed the gel inside the machine, closed the door,
and pressed on the “transilluminator” button to turn on the UV light. Under the UV light,
the ethidium bromide lighted up and clearly showed the bright white bands of the
standard. The four DNA bands showed up in a grayish color.
I noticed that the left side of the gel, i.e. the side of the wells, was much lighter than the
other side. Dr. Ju helped me to figure out that since the ethidium in ethidium bromide is
positive, it goes to the negative side of the current, which is the well side (opposite to the
direction of DNA).
Then Dr. Ju put the gel on an older UV light machine that has a transparent protective
cover. The ethidium bromide showed up as red bands.
From 12:00pm – 1:00pm all the SEAP students in the Rickettsia department attended a
meeting with other scientists and Commander Fryauff in the department. We each talked
about our projects and what we have done this week. I printed out my logs and said that
my project is to select and optimize the probe to detect the flagellin gene of Borrelia
lonestari.
Dr. Ju had to leave early because of a family issue so I wrote my log in the afternoon.
3. Reflections
a. actually accomplished: worked with Clustal W program and Gene Doc, practiced gel
loading, observed ethidium bromide under UV light machines, and talked about my
project at the meeting .
b. concerns: none for today 
c. learning: I was glad that I finally understood the difference between the DNA dye and
ethidium bromide. The DNA dye is just an indicator for the scientist to know that the
bands are not running out of the gel.
I was also happy about observing the ethidium bromide results for the first time in my life
(never done it in DNA I, because ethidium bromide is dangerous).
4. Planning
My goal for the next day is to carry out the PCR process by myself successfully and start
working with qPCR.